In the semiconductor industry there has been an ongoing trend towards smaller and smaller electronic circuits and devices. This technology has been sustained by modifying the capabilities of manufacturing processes such as photolithography. Such top-bottom manufacturing methods are reaching their limits in attempts to develop smaller feature sizes. Implementation of a bottom-up method is now sought after for the manufacture of nanoscale electronic circuits. A bottom-up method utilizes the self-assembly nature of biological structures for the formation of structures from atomic or molecular constituents. Control of interconnections emerges as one of the major challenges in the development of these bottom-up approaches. Research suggests that proteins and assemblies of proteins offer the control necessary for inexpensive and reliable fabrication of nanoscale interconnects. One approach to the fabrication of interconnects for semiconductor application has involved using biological molecules as templates for metallization.
Microtubules (MT) are naturally formed tubular structures, 25 nm in outer diameter with inner diameter of 15 nm and lengths of several micrometers (Schuyler, S. C.; Pellman, D. Microtubule ‘plus-end-tracking proteins’: the end is just the beginning. Cell 2001, 105(4), 421-424). MTs are biopolymers assembled from protein heterodimers containing both alpha- and beta-tubulin (see FIG. 1). In the presence of the small molecule guanosine triphosphate (GTP), the tubulin heterodimer (Tu-GTP) self-assembles into the MT structure. The MTs' aspect ratio, chemical polarity, reversibility in assembly and ability to be metalized by electroless plating (2-3) make them good candidates to serve as templates for the fabrication of nanoscale systems, including those based on metallic nanowires (Mertig, M.; Kirsch, R.; Pompe, W. Biomolecular approach to nanotube fabrication. Applied Physics A. 1998, 66, S723-S727). In addition, microtubules can provide biological interactions with a native high specificity (Whaley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F. & Belcher, A. M. Selection of peptides with semiconductor binding specificity for directed nanocrystal assembly. Nature 2000, 405, 665-668; Sarikaya, M.; Tamerler, C.; Jen, A. K. Molecular biomimetics: nanotechnology through biology. Nature Materials 2003, 2(9), 577-585; Antikainen, N. M.; Martin, S. F. Altering protein specificity: techniques and applications. Bioorganic & Medicinal Chemistry 2005, 13(8), 2701-2716). The exposure of different tubulin regions at either end of a microtubule (the plus or minus ends) makes it possible to control MT attachment to substrates in a specific orientation.
For example, Limberis et al. took advantage of the polarity and specificity of biological interactions of MTs to flow-align pre-grown MTs immobilized onto a silica substrate using a single-chain antibody that binds only to a portion of α-tubulin exposed at the MT minus end (Limberis, L.; Magda, J. J.; Stewart, R. J. Polarized Alignment and Surface Immobilization of Microtubules for Kinesin-Powered Nanodevices. Nano Lett. 2001, 1(5), 277-280).
MTs are polarized with a slow-growing end (the so-called minus end exposing α-tubulin) and fast-growing end (the β-tubulin terminated plus end). The plus end of a MT typically grows at a rate 5 to 10 times faster than the minus end. In vitro, MTs can be grown from solutions containing high concentrations of purified tubulin (Johnson, K. A.; Borisy, G. G. Kinetic analysis of microtubule self-assembly in vitro. J Mol. Biol. 1977, 117, 1-31; Bayley, P. M.; Martin, S. R. Inhibition of microtubule elongation by GDP. Biochemical and Biophysical Research Communications 1986, 137(1), 351-358; M. Caplow, J. Shanks, S. Breidenbach, R. L. Ruhlen, Kinetics and mechanism of microtubule length changes by dynamic instability. J. Biol. Chem. 1988, 263(22), 10943-10951; Simon, J. R.; Salmon, E. D. The structure of microtubule ends during the elongation and shortening phases of dynamic instability examined by negative-stain electron microscopy. J. Cell Sci. 1990, 96(4), 571-582; Kowalski, R. J.; Williams, R. C. Jr. Microtubule-associated protein 2 alters the dynamic properties of microtubule assembly and disassembly. J. Biol. Chem. 1993, 268(13), 9847-9855; Marx, A.; Mandelkow, E. A model of microtubule oscillations. European Biophysics Journal 1994, 22(6), 405-421; Caudron, N.; Valiron, O.; Usson, Y.; Valiron, P.; Job, D. A reassessment of the factors affecting microtubule assembly and disassembly in vitro. Journal of Molecular Biology 2000, 297(1), 211-220). Microtubules generated from pure tubulin exist in a dynamic state (called dynamic instability) with net addition of tubulin to the plus end and net removal of tubulin from the minus end. This “treadmilling” effect can be controlled via interaction of the MT with various chemical agents (i.e. microtubule associated proteins (MAP), taxol) resulting in relatively stable MTs (Kinoshita, K.; Arnal, I.; Desai, A.; Drechsel, D. N.; Hyman, A. A. Reconstitution of physiological microtubule dynamics using purified components. Science 2001, 294, 1340-1343; Arnal, I.; Wade, R. H. How does taxol stabilize microtubules? Current Biology 1995, 5, 900-908).
In the absence of these agents and for tubulin concentrations below a critical value, Cc, MTs will depolymerize (Lodish, H.; Berk, A.; Zipuski, S. L.; Matsudaira, P.; Baltimore, D. and Darnell, J. “Molecular Cell Biology,” 4th Edition, Freeman, 2000.) Tubulin dimers polymerize into MTs for tubulin concentrations above Cc. At concentrations of tubulin dimers near Cc, individual MTs exhibit dynamic instability (Mitchison, T. and Kirschner, M. Microtubule assembly nucleated by isolated centrosomes. Nature 1984, 312, 237-242.) and undergo apparently random successive periods of disassembly (catastrophe) and assembly (rescue). The mechanism for transition between a growing state and a shrinking state is generally believed to be associated with hydrolysis of bound GTP when tubulin heterodimers become incorporated within the microtubule structure. While the process of MT growth is reasonably well understood, in vivo and in vitro MT nucleation is, however, still poorly understood. Within the cell, the minus end is tethered to microtubule-organizing centers (MTOC) such as centrosomes, and the plus end extends into the cytoplasm (Job, D.; Valiron, O.; Oakley, B. Microtubule nucleation. Current Opinion in Cell Biology 2003, 15(1), 111-117). MT assembly is believed to nucleate from the MTOC through interaction with a tubulin isoform, gamma-tubulin (Moritz, M.; Zheng, Y.; Alberts, B. M.; Oegema, K. Recruitment of the gamma-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds. J Cell Biol. 1998, 142, 775-786; Schnackenberg, B. J.; Khodjakov, A.; Rieder, C. L.; Palazzo, R. E. The disassembly and reassembly of functional centrosomes in vitro. Proc Natl Acad Sci USA 1998, 95, 9295-3900; Gunawardane, R. N.; Lizarraga, S. B.; Wiese, C.; Wilde, A.; Zheng, Y. Gamma-Tubulin complexes and their role in microtubule nucleation. Curr. Top Dev. Biol. 2000, 49, 55-73). Research in vitro has shown that gamma-tubulin is an essential component in the centrosome for microtubule nucleation (Felix, M. A.; Antony, C.; Wright, M.; Maro, B. Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation. J. Cell Biol. 1994, 124, 19-31; Stearns, T.; Kirschner, M. In vitro reconstitution of centrosome assembly and function: the central role of gamma-tubulin. Cell 1994, 76, 623-637).
Monomeric gamma-tubulin and gamma-tubulin protein complexes can both nucleate MT. The nucleation time of MTs has been shown to be shorter in the presence of monomeric gamma-tubulin (Leguy, R.; Melki, R.; Pantaloni, D.; Carlier, M. F. Monomeric gamma-tubulin nucleates microtubules. J. Bio. Chem. 2000, 275(29), 21975-21980). In vitro, monomeric gamma-tubulin behaves as a minus-end-specific protein, with very high binding specificity to the microtubule end. It caps microtubule minus ends and catalyzes microtubule nucleation (Leguy, R.; Melki, R.; Pantaloni, D.; Carlier, M. F. Monomeric gamma-tubulin nucleates microtubules. J. Bio. Chem. 2000, 275(29), 21975-21980; Li, Q.; Joshi, H. C.; Gamma-tubulin is a minus end-specific microtubule binding protein. J. Cell Biol. 1995, 131, 207-214). Specific peptides and/or complexes of gamma-tubulin have also been identified to serve as binding sites to interact with tubulin heterodimers (Llanos, R.; Chevrier, V.; Ronjat, M.; Meurer-Grob, P.; Martinez, P.; Frank, R.; Bornens, M.; Wade, R. H.; Wehland, J.; Job, D. Tubulin binding sites on gamma-tubulin: identification and molecular characterization. Biochemistry 1999, 38, 15712-15720; Fuller, S. D.; Gowen, B. E.; Reinsch, S.; Sawyer, A.; Buendia, B.; Wepf, R.; Karsenti, E. The core of the mammalian centriole contains gamma-tubulin. Curr Biol. 1995, 5(12), 1384-1393; Moritz, M.; Braunfeld, M. B.; Sedat, J. W.; Alberts, B.; Agard, D. A. Microtubule nucleation by g-tubulin-containing rings in the centrosome. Nature (London), 1995, 378(6557), 638-640; Wiese, C.; Zheng, Y. A new function for the g-tubulin complex as a microtubule minus-end cap. Nature Cell Biology 2000, 3, 358-364; Oegema, K.; Wiese, C.; Martin, O. C.; Milligan, R. A.; Iwamatsu, A.; Mitchison, T. J.; Zheng, Y. Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules. J. Cell Biol. 1999, 144, 721-733). Gamma-tubulin ring complex (gamma-TuRC), which also binds to the minus ends of microtubules, can also work as a nucleation center for growth of the microtubule both in vivo and in vitro (Fuller, S. D.; Gowen, B. E.; Reinsch, S.; Sawyer, A.; Buendia, B.; Wepf, R.; Karsenti, E. The core of the mammalian centriole contains gamma-tubulin. Curr Biol. 1995, 5(12), 1384-1393; Moritz, M.; Braunfeld, M. B.; Sedat, J. W.; Alberts, B.; Agard, D. A. Microtubule nucleation by g-tubulin-containing rings in the centrosome. Nature (London), 1995, 378(6557), 638-640; Wiese, C.; Zheng, Y. A new function for the g-tubulin complex as a microtubule minus-end cap. Nature Cell Biology 2000, 3, 358-364). In addition to gamma-TuRC, several smaller gamma-tubulin complexes, called gamma-tubulin small complexes (gamma-TuSCs) are identified as components of gamma-TuRC(32). Gamma-TuSCs can also nucleate microtubule in tubulin solutions but with lower efficiency compared with gamma-TuRCs(32). Besides growing from centrosomal sites, MTs also can grow from noncentrosomal sites in the cell. In the absence of a centrosome, other mechanisms must operate to organize free MTs. One such mechanism is self-organization, which can produce MT asters, bundles, and bipolar spindles. It has been shown that microtubules can also grow from some small chromatin-coated beads (Heald, R.; Tournebize, R.; Blank, T.; Sandaltzopoulos, R.; Becker, P.; Hyman, A.; Karsenti, E. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 1996, 382, 420-425).
This invention relates to the use of microtubules (MT) to form 2D- and 3D-structures on, between and among substrates. Creation of such structures relies on in-situ growth of MTs from selected nucleation sites on substrates and the capture of growing ends (+ends) of the MTs at selected capture sites separated from the nucleation sites. These structures can be generally used as nanoscale templates, as scaffolds for attachment and location of nanoscale objects. More specifically the can be used as templates for fabricating nanoscale interconnects, interconnect arrays, and networks. The ability to create and use arrays or structures of MTs, particularly as templates for interconnecting devices on microchips, necessitates the development of a protocol where MTs can be nucleated and directionally grown from specific sites on the microchip toward some target capture site elsewhere on that chip. As a step in the process of manufacturing MT-based nanostructures on a silicon wafer, this invention provides an “in situ” approach to forming MT-based nanostructures comprising functionalizing selected different sites on a substrate (e.g., a metal pad) with derivatized MT nucleating complexes and derivatized MT capture complexes, followed by surface-driven growth of MTs from nucleating sites followed by capture of growing MTs at capture sites to form an MT structural link between two selected sites on a substrate. The advantage of this approach lies not only in the immobilization of MTs on the surface of a substrate, but more importantly on the unique ability to initiate MT growth from selected sites and the ability to generate MT's between selected sites.
Another requirement in the manufacture of MT-based nanostructures for forming electrical circuits and devices is the development of improved metallization techniques. Several biological templates have already been shown to form nanostructures through metallization processes. In a study by Braun et al., DNA is used as a template for creating a 12 μm long and 100 nm wide silver wire. This was accomplished by first fixing the DNA between gold electrodes followed by selective localization of silver ions along the DNA skeleton; the silver-ions were then reduced to silver metal aggregates along the DNA to yield a nanowire which exhibited granular morphology and the ability to conduct electrical current [E. Braun, Y. Eichen, U. Sivan, G. Ben-Yoseph, Nature 1998, 391, 775.]. Similarly, DNA has also been metalized with nanoscale palladium clusters. The DNA was activated with Pd ions and then added to a reduction bath containing dimethylamine borane (DMAB) as the reducing agent. Over time the initial clusters that formed on the surface become a continuous metallic surface [J. Richter et al, Adv. Mater. 2000, 12(7), 507].
Others have used viruses, which are essentially helical RNA, as the substrate for the plating of nickel and cobalt metal. A tubular virus, tobacco mosaic virus, was metalized on the inner and outer surfaces. This selective metallization was regulated by the absence or presence of phosphate that interacted with functional groups that differed on the inside and outside surfaces [M. Knez et al, Nano Letters 2003, 3(8), 1079 and M. Knez et al, Adv. Funct. Mater. 2004, 14(2), 116].
Microtubules have also been coated by electroless deposition of nickel and cobalt. Electroless deposition is a redox reaction, in which a cation of a metal is chemically reduced onto a surface to form a metal film. Typically, the metallization of MTs involves a two step process of activating the MT surface with a noble metal such as Pd or Pt, which is a catalyst for the electroless deposition of the desired metal. Nickel plating of MTs was carried out under physiological conditions, between 30-60° C. and between pH 6 and 8. The metallization process produced nickel only in areas where the Pd catalysts were deposited. While Pd and Pt ions have the capability to diffuse through the MT wall, no deposition was observed on the inner channel due to the rapid metal deposition on the outer surface which blocked ion penetration. Nickel nanowires generated had an overall diameter of 50 to 60 nm. Similar results were reported for cobalt metallization [R. Kirsch, M. Mertig, W. Pompe, R. Wahl, G. Sadowski, K. J. Böhm, and E. Unger, Thin Solid Films 1997, 305, 248; M. Mertig, R. Kirsch, W. Pompe, Applied Physics A 1998, 66, S723.]. MTs have also been metalized with Pd which was proposed to proceed by binding of Pd particles with histidine amino acids on the surface. The surface of the MTs was covered with palladium particles of 2 to 3 nm to form quasi-continuous coverage up to 100 nm in diameter [S. Behrens, K. Rahn, W. Habicht, K. J. Böhm, H. Rösner, E. Dinjus, E. Unger Adv. Mater. 2002, 14(22), 1621].
U.S. published patent application 20040063915 relates to metallization of MTs by reacting “fixed” microtubules with a reducible metal salt. MTs are fixed by treatment with glutaraldehyde. Noble metal salts, e.g., HAuCl4, in combination with a reducing agent (NaBH4 or sodium ascorbate) are used to metalize fixed MTs. Additional salts are said to be useful in the method, including AgNO3, HPtCl3, CuNO3, and K2PdCl4.
Copper metallization of templates to produce nanostructures is of particular interest to the semiconductor industry, because copper is currently the interconnect metal of choice in integrated circuits. Copper is a more desired metal than nickel or cobalt due to its lower resistivity. U.S. published patent application 20040063915 suggests that MTs can be metalized employing CuNO3 as a reducible salt, but does not demonstrate copper metallization of MTS. Furthermore, the published application requires fixing of MTs prior to metallization. Copper plating on bolaamphiphile nanotubes [H. Matsui, S. Pan, B. Gologan, and S. H. Jonas, J. Phys. Chem. B 2000, 104, 9576] has been reported. Bolaamphiphiles are self-assembling, organic structures in the form of a crystalline tubule with an average diameter of 700 nm and a length of 10 μm. Metallization with copper and nickel was carried out by exposure to an electroless nickel or copper bath with the reducing agents hypophosphite and dimethylamineborane (DMAB), respectively. The copper coated nanotubes had a diameter of 700 nm and the nickel coated tubes had a diameter of 1 μm. Metallization occurred with and without the initial activation of the surface with a Pd catalyst. The metallization was reported to result from the binding of ions to available amine groups in the peptide followed by reduction of these ions to metal in the plating baths.
Electroless deposition of copper onto the MT surface poses a challenge, because the commercially available plating baths contain formaldehyde which damage or destroy MTs. Conditions typically employed for electroless deposition of copper are very harsh—alkaline pH values (11.5 to 13) and temperatures from 55° C. to 70° C. which are detrimental to MTs [Y. Shacham-Diamand, J. Micromech. Microengr., 1991, 1, 66].
The present invention provides an improved method for metallization of MTs and other biological templates by electroless deposition of copper and other metals onto MTs. The method employs reducible salts, such as CuSO4, in the presence of a reducing agent at pH of 4 or less which is not detrimental to MT function and structure. Furthermore, the metallization method is compatible with the methods herein for growth of MTs structures on substrates. Additionally, the method has been found to be useful for forming metalized MTs having diameters of 15 nm or more. In order to obtain metalized MTs of such small diameters, it is believed that metallization of MTs with Cu at least proceeds through deposition of metal inside of the MTs.